MATERNAL AND NEONATAL EVALUATION OF REACTIVE OXIGEN 1
METABOLITES (D-ROMS) AND BIOLOGICAL ANTIOXIDANT POTENTIAL (BAP) IN 2
THE HORSE 3
Sgorbini M., Bonelli F., Rota A., Marmorini P., Biagi G., Corazza M., Pasquini A. 4
Department of Veterinary Sciences, University of Pisa, via Livornese snc, 56122, San Piero a 5
Grado, Pisa, Italy. 6
Corresponding author: 7
Micaela Sgorbini 8
Department of Veterinary Sciences 9
via Livornese snc, 56122, San Piero a Grado, Pisa, Italy 10 Phone: +390502210117 11 Fax: +390502210182 12 sgorbini@vet.unipi.it 13
Authors’ e-mail addresses: f.bonelli@vet.unipi.it; alerota@vet.unipi.it; ninnaro@tiscali.it; 14
giulia.biagi@vet.unipi.it; michele.corazza@unipi.it; anna.pasquini@vet.unipi.it 15
16
Abstract 17
The aim of the present work was to evaluate Reactive Oxygen Metabolites and biological 18
antioxidant potential in mares and foals in order to study perinatal oxidative status. A total of 60 19
animals were included in the present study. Maternal and foal venous blood samples were collected 20
immediately after delivery along with a sample drawn from one of the umbilical arteries and plasma 21
samples were evaluated for lactatemia, d-ROMs and BAP. T test for unpaired data was applied 22
between mares vs umbilical artery blood vs foals, both for d-ROMs and BAP. Pearson test with 23
two-tailed p-value and a confidence interval of 95% was performed between d-ROMs and BAP, and 24
between d-ROMs and lactatemia, both for mares and foals. Finally, T-test for unpaired data was 25
performed between fillies and colts. T-test showed differences between mares vs their own foals vs 26
umbilical artery blood, but not foals vs umbilical artery blood, both for d-ROMs and BAP. A 27
positive correlation was found both in mares and foals between BAP and d-ROMs, and in mares 28
between lactatemia and d-ROM. No differences in gender were found in BAP concentration. Our 29
data are in line to previous studies performed in women and cattle. 30
31
Key words 32
Mare, foal, oxidative stress, BAP, d-ROMs 33
35
1. Introduction 36
Oxidative stress reflects an imbalance between the systemic manifestation of reactive oxygen 37
species and a biological system's ability to readily detoxify the reactive intermediates or to repair 38
the resulting damage. [1-5] During pregnancy, delivery and lactation, an overproduction of reactive 39
oxygen species (ROS) may take place due to the increased metabolism, along with the physical 40
effort. [6-8] Several papers confirmed the presence of oxidative stress during pregnancy, [9] 41
parturition, [10-11] post-parturient period in women. [12-14] and also newborn babies are 42
susceptible to oxidative stress due to an imbalance between antioxidant and oxidant-generating 43
systems. [15-26] The placenta seems to have an important role in protecting human fetus from 44
oxidative stress. In fact, many clinical studies reported the protective mechanism against O2 toxicity 45
in the human feto-placental unit during pregnancy and at time of parturition. [5, 26-28] In 46
veterinary medicine, antioxidative/oxidative status has been studied during pregnancy, lactation, 47
and at delivery both in cows and their newborn calves and ewes [29-34] The aim of the present 48
work was to measure mares and foals concentrations of reactive oxygen metabolites (d-ROMs) and 49
biological antioxidant potential (BAP) in order to evaluate maternal and neonatal oxidative status at 50
delivery and to verify the protective role of placenta against fetal oxidative stress. 51
52
2. Materials and Methods 53
2.1 Animals 54
Two Throughbred, 1 Purebred Arabian, 27 Standardbred trotter, 4 mixed breed mares and their 55
foals, for a total of 68 animals housed at the studfarm “La Piaggia” (Galleno, Pisa, Italy) and 56
admitted at the Department of Veterinary Sciences, University of Pisa from 2012 to 2013 foaling 57
seasons were included in the present study. The mares were hospitalized because the owners 58
requested an attended parturition. Ethical approval and informed written consent were obtained 59
from the owners according to the Ethics Committee on Animal Experimentation of the University 60
of Pisa. Mares and their foals underwent similar management conditions. Mares were included in 61
this study according to the following criteria: 1) pregnancy length > 320 days; [35] 2) unassisted 62
delivery; 3) mares treated against gastrointestinal parasites and vaccinated against equine influenza, 63
tetanus, and equine herpes virus-1 according to guidelines of the American Association of Equine 64
Practitioners; [36] 4) healthy at physical examination. Attendance at birth was ensured by 65
monitoring mammary development and measuring the concentration of calcium in the mare's 66
colostrum every evening at 6 pm using a commercially avalilable kit (Foal WatchTM, Chemetrics, 67
Calverton, VA, USA). Mares were constantly supervised 24 hours/day when calcium in the 68
colostrum was over 200 ppm. 69
Inclusion criteria for foals were: 1) Apgar Score 5 minutes after birth ≥7; [37] 2) IgG ≥ 800 mg/dl at 70
24 hours of age (Snap Foal IgG test Kit, Idexx, USA); [37] 3) righting reflex present immediately 71
after foaling, suck reflex within 10 minutes, sternal recumbence within 5 minutes, quadrupedal 72
position within 60 minutes and nursing the mare within 120 minutes after birth. [38] Foals were 73
given physical examinations before each blood collection and appeared to be clinically healthy 74
during the all study period. 75
76
2.2. Sample collection and handling 77
Immediately after delivery, maternal and foal venous blood samples were drawn by the jugular vein 78
and collected into lithium-heparinized test tubes (FL Medical, Padua, Italy). Simultaneously a 79
heparinized blood sample was drawn from one of the umbilical arteries from each umbilical cord. 80
Heparinised samples were centrifuged at 3000 g for 10 minutes, as recommended by the 81
manufacturer, plasma was frozen at -18 °C and then analysed in a single batch. 82
83
2.3. Biochemical analysis 84
All plasma samples were evaluated for plasma lactate (Accutrend LactateÒ, Micralab srl, MI), 85
plasma reactive oxygen metabolites concentrations by d-ROMs test (Diacron srl, Grosseto, Italy) 86
and biological antioxidant potential by BAP test (Diacron srl, Grosseto, Italy). D-ROMs and BAP 87
tests were carried out by a spectrophotometer (Slim, SEAC, Florence, Italy). The stability of the d-88
ROMs and BAP tests on stored horse blood was previously evaluated. [39] 89
2.4 Statistical analysis 91
The maternal, umbilical artery blood and neonatal d-ROMs and BAP concentrations, and lactatemia 92
were expressed as mean±standard deviation (X±SD). Normal distribution of data was evaluated by 93
Komolgorov-Smirnov test. Data showed a Gaussian distribution. T test for unpaired data was 94
applied to verify differences between mares and their own umbilical artery blood and foals. Pearson 95
test with two-tailed p-value and a confidence interval of 95% was performed to evaluate correlation 96
between d-ROMs and BAP, and between d-ROMs and lactatemia, both for mares and foals. Finally, 97
T-test for unpaired data was performed between fillies (n=16) vs colts (n=18) to verify differences 98
in antioxidant status related to gender. Results were considered to be statistically significant when 99
p<0.05 (Statgraphics plus, USA). 100
101
3. Results 102
d-ROMs and BAP concentrations in mares, umbilical artery blood and foals are reported in Table 1. 103
Lactatemia in mares and foals was 2.96±1.15mmol/L and 4.01±1.71 mmol/L, respectively. T-test 104
for unpaired data showed statistical differences between mares vs their own foals (p<0.0001), mares 105
vs umbilical artery blood (p=0.005), but not foals vs umbilical artery blood (p=0.3) for d-ROMs. 106
BAP concentration was statistically different between mares vs foals (p=0.04) and mares vs 107
umbilical artery blood (p=0.02), but not between foals vs umbilical artery blood (p=0.7). Pearson 108
test showed a positive correlation in mares between BAP and d-ROMs (r2= 0.644, p=0.0001) and 109
between lactatemia and d-ROMs (r2=0.561, p<0.001), while in foals correlation was found only 110
between d-ROMs and BAP (r2=0.653, p<0.01). T-test performed between fillies and colts did not 111
show statistical differences for d-ROMs and BAP concentrations related to gender (p=0.3). 112
113
4. Discussions and conclusions 114
Oxidative stress is defined as an imbalance between oxidants and antioxidants in which the oxidant 115
activity exceeds the neutralizing capability of antioxidants, resulting in cellular injury and activation 116
of pathologic pathways. [2] This work investigates antioxidative⁄oxidative profile of mares and their 117
own foals measuring the d-ROMs and BAP concentrations. 118
In this study, the d-ROMs amount was higher in maternal blood than in umbilical artery blood and 119
foals’ blood at birth. In women the concentration of lipoperoxides increases as pregnancy advances 120
and the highest concentrations are observed during delivery. [5] Moreover, the concentration of 121
oxidative agents is higher in maternal blood than in cord blood at birth. [40] In our study, the 122
concentration of d-ROMs has not been determined during pregnancy, but our results regarding the 123
differences in d-ROMs concentration between mares and umbilical artery blood/foals at birth are in 124
line to previous studies performed in women. [5, 40] These differences could support the hypothesis 125
of the positive role of the placenta in preventing the passage of oxidative agents from the mare to 126
the foal at the time of parturition, as suggested in humans. [5, 27] The prevention could be due to a 127
higher placenta secretion of oxidative agents at the maternal side compared to the foetal side, as 128
suggested for women. [41-42] 129
In the present study, also BAP concentration in the mare is higher than in the umbilical artery blood 130
and in the foal. Newborn babies are at high risk for oxidative stress at birth and are very susceptible 131
to oxidative damage by Reactive Oxigen Substances (ROS) because the extrauterine environment is 132
richer in oxygen than the intrauterine environment. During delivery, in fact, the foetus is transferred 133
from an intrauterine hypoxic environment with 20–25 mmHg oxygen tension (paO2: 20–25 mmHg) 134
to an extrauterine normoxic environment with approximately 100 mmHg paO2. [43] The increase in 135
oxygen tension induces the production of ROS [44-45] that is also exacerbated by the low 136
efficiency of the natural antioxidant system in newborns. [5, 21, 46] Inami et al. [47] reported lower 137
antioxidative activities and higher concentration of thiobarbituric acid-reactive substances in serum 138
of newborn calves as their dams. Our results on BAP values are in line with literature; the lower 139
BAP concentration in foals than in mares obtained in this study may be caused to a lower 140
antioxidative activity in newborn foals due to immature defence systems, as already reported for 141
babies and cows. [31, 47] 142
Castillo et al. [29] studied antioxidative/oxidative status of plasma in cows from 10 weeks before 143
parturition until 8 weeks post-partum. The authors observed an increase in antioxidant status up to 1 144
week postpartum and then a decrease till 8 weeks postpartum associated to a parallel peroxidative 145
damage trend. Our results reported a positive correlation (r= 0.644 p<0.05) between BAP 146
antioxidants and oxidative parameters in the mare, similarly to what happens in cattle, suggesting 147
an effective reaction of the antioxidant system in healthy mares during critical period, such as 148
parturition and lactation. [29, 31, 48] The missing correlation between d-ROMs and BAP amounts 149
in foals could be explained by the lower antioxidant activity in newborn in comparison with adult 150
horses. [31, 47] 151
Lista et al. [5] reported the influence of gender on oxidative/antioxidative status. The authors 152
observed that females were less susceptible to oxidative stress because they showed higher values 153
of total antioxidative system as compared to males. To the authors’ knowledge there are no data on 154
this item in veterinary medicine. Our results did not show differences on BAP concentration 155
between fillies and colts, thus in foals the antioxidative activity did not seem to be influenced by 156
gender. 157
The most common cause of an increase in blood lactate concentration in horses is a decreased tissue 158
perfusion and oxygen delivery with subsequent anaerobic metabolism. In adult horses, decreased 159
tissue perfusion is most often due to hypovolemia, but inappropriate vascular tone during severe 160
sepsis may also contribute to hyperlactatemia. In septic equine neonates, hypovolemia, 161
inappropriate vascular tone, and decreased cardiac output have been implicated as causes of 162
hyperlactatemia. [49-51] Hypoxia-reperfusion and inflammation are among the major mechanisms 163
of induction of oxidative stress. [52] Besides, lactate induces the production of mitocondrial 164
reactive oxigen species (ROS). [53] Our results show a positive correlation between lactatemia and 165
d-ROMs in mares at parturition, suggesting that the rise of d-ROMs at delivery may be due to the 166
increase of blood lactate. 167
Our results are in line with literature in humans and other animal species, confirming the 168
importance of a balanced oxidative status in mares at delivery. 169
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289
Maternal blood Cord blood Foal blood d-ROMs (U.Carr.) 188.5±62.4a 142.9±60.0b 127.4±45.6b BAP μmol/l 2064.0±459.7c 1780.0±479.3d 1821.0±451d
Table 1. d-ROMs and BAP concentrations in mares, umbilical cord and foals expressed as 290
mean±standard deviation. Within row, different superscripts denote a significant difference (a≠b; 291
c≠d) (p<0.05). 292
